title: Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria.
abstract: Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-beta-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.
authors.affiliation: Instituto Leônidas e Maria Deane-Fiocruz, Rua Teresina 476, 69057-070 Manaus, AM, Brasil.
authors.forename: Luis Andre
authors.initials: LA
authors.lastname: Mariuba
authors.forename: Patricia Puccinelli
authors.initials: PP
authors.lastname: Orlandi
authors.forename: Márcia
authors.initials: M
authors.lastname: Medeiros
authors.forename: Rudson
authors.initials: R
authors.lastname: Holanda
authors.forename: Andrea
authors.initials: A
authors.lastname: Grava
authors.forename: Paulo Afonso
authors.initials: PA
authors.lastname: Nogueira
mesh_terms: D000740:Anemia / Q000276:immunology / Q000469:parasitology*; D000818:Animals; D001483:Base Sequence; D016778:Malaria, Falciparum / Q000150:complications* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000235:genetics / Q000276:immunology*; D011489:Protein Denaturation; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins; C073168:rhoptry-associated antigen-2, Plasmodium